Revealing Secrets of Cellular Phosphorylation Systems Using Mass Spectrometry

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Protein phosphorylation is a chemical modification utilized by all living organisms to alter the function of proteins and controls processes such as cellular communication, growth, metabolism, differentiation, and survival. The enzymes that add and remove phosphate molecules to proteins are called kinases and phosphatases respectively.  Since a delicate balance of reversible phosphorylation is essential for cellular homeostasis, the actions of all kinases/phosphatases are under tight surveillance. One of the most powerful analytical tools to study phosphorylation systems is biological mass spectrometry. Mass spectrometry based methods can rapidly identify functional multiprotein complexes containing kinases and phosphatases as well as directly map the location of phosphate addition to better study the consequence of the protein modification. Accordingly, the long term goal of our research program is to integrate mass spectrometry, biochemistry, and cell biology techniques to decipher the mechanistic details regulating poorly understood kinases and phosphatases predicted to regulate fundamental cellular processes. ***     Currently we are directing much of our focus on a poorly understood phosphatase identified as YVH1 (also known as DUSP12). Human YVH1 (hYVH1) is a so called dual specificity phosphatase (DSP) widely expressed in human tissues. The YVH1 genes are found throughout the animal and plant kingdom and all gene products possess a characteristic zinc binding domain in addition to their phosphatase domain. Remarkably, although YVH1 was the first eukaryotic DSP identified over 22 years ago, relatively little is known about its specific biological function in human cells. ***    The recent acquisition in 2014 of a new multiplexed mass spectrometry system capable of biological applications such as discovery/targeted proteomics, mapping of post translational modifications, structural protein dynamics, and interactome analysis has fundamentally altered the depth of analysis for our research program. Utilizing this system, we have recently isolated novel hYVH1 associating proteins from human cell lines using classical mass spectrometry based affinity chromatography techniques. Our preliminary findings suggest that hYVH1 is active at cellular components that control the rate at which genes are transformed into functional proteins (mRNA regulation). We are now interested in employing next generation affinity capture methods and magnetic bead technology to thoroughly delineate and functionally validate the hYVH1 interactome. Follow-up experiments will include examining how the cell controls the access of hYVH1 to these macromolecular workstations.  The methods employed within our proposal will be transferrable to the study of other poorly understood phosphatases while the findings will deepen our understanding of the role DSPs play in cellular regulation.